Stable intermediates can be trapped during the reversible refolding of urea-denatured rhodanese.
The enzyme rhodanese (EC 2.8.1.1) could be reversibly refolded from urea in the presence of lauryl maltoside, beta-mercaptoethanol, and sodium thiosulfate. The unfolding/folding transition monitored using intrinsic fluorescence was resolved into two two-state transitions with midpoints at 3.6 and 5.0 M urea. The analysis assumed an intermediate with an emission maximum at 345 nm. Monitoring anisotropy of intrinsic fluorescence also gave an asymmetric transition. Activity followed one two-state transition centered at 3.6 M urea with no major change of secondary structure. Without thiosulfate or mercaptoethanol, there was one two-state transition at 5.0 M urea giving a species, in dilute urea, with a fluorescence maximum at 345 nm. This intermediate slowly relaxed toward 335 nm (t1/2 = 85 min) if only thiosulfate was absent but without regaining activity. Subsequent addition of thiosulfate led to a first-order recovery of activity (t1/2 = 75 min). Thus, a possible folding intermediate can be trapped which displays increased access of water and solutes to its fluorescent tryptophans. This intermediate conformer, which is flexible, has considerable secondary structure, is inactive, has exposed hydrophobic surfaces, and requires specific reducing conditions to regain full activity. Refolding probably involves an initial, rapid, hydrophobic collapse with acquisition of secondary structure to form the intermediate, followed by slower adjustment to the native global conformation. Final reactivation requires reduction localized at the active site.
Horowitz
P M
PM
University of Texas Health Science Center, Department of Biochemistry, San Antonio 78284-7760.
Criscimagna
N L
NL
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013466:Sulfurtransferases / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*; D013997:Time Factors; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology*
D014364:Tryptophan; D014508:Urea; D004228:Dithionitrobenzoic Acid; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase